Romanian Biotechnological Letters Vol. , No. x,

Copyright © 2017 University of Bucharest Printed in Romania. All rights reserved

ORIGINAL PAPER

Clonal propagation, antioxidant activity and phenolic profiles of

Convolvulus galaticus Rostan ex Choisy

Received for publication, December, 27, 2015

Accepted, November, 8, 2016

ARZU UCAR TURKER

1*, ARZU BIRINCI YILDIRIM

2

1Abant Izzet Baysal University, Department of Biology, Faculty of Science and Art, Bolu,

Turkey 2Abant Izzet Baysal University, Department of Field Crops, Faculty of Agricultural and

Environmental Science, Bolu, Turkey

*Address for correspondence to: turker_a@ibu.edu.tr

Abstract

Convolvulus galaticus Rostan ex Choisy (grizzle bindweed) is a medicinal plant in the family

Convolvulaceae. The first objective of this study was to determine a highly efficient and rapid

regeneration system for C. galaticus. Secondly, field-grown and in vitro-grown plants were compared

in terms of antioxidant activities and phenolic constituents. C. galaticus leaves and stems were surface

sterilized and three different explants (leaf, stem and node) were cultured. Regeneration was observed

only with node explants. Best shoot proliferation was observed with 0.5 mg/l TDZ and 1.0 mg/l IAA,

producing 15 shoots per explant at 84 % frequency. In vitro regenerated plants were also used as

donor plants for explant source and best shoot formation was observed with 1.0 mg/l TDZ and 0.5 mg/l

IBA with node explant, producing 23.7 shoots per explant at 60 % frequency. Regenerated shoots were

transferred to rooting media and 1.0 mg/l IBA was the most effective for rooting. In the second part of

this study, methanolic extract of field-grown and in vitro-grown C. galaticus were compared in terms

of antioxidant activity and phenolic constituents. Field-grown plant showed higher antioxidant

activities and phenolic content than in vitro-grown plant.

Keywords: antioxidant, Convolvulus galaticus, in vitro culture, LC-MS/MS, phenolics

1. Introduction Convolvulus galaticus Rostan ex Choisy (Grizzle bindweed) is an endemic, prostrate,

herbaceous, perennial herb in the family Convolvulaceae (DAVIS [1]). The natural habitat

for C. galaticus is Pinus woods, open steppe, stony slopes, meadows, cultivated and fallow

fields which are usually calcareous at 880-2000 m. It is found in the inner and rarely in the

North part of Turkey (DAVIS [1]). According to some ethnobotanical studies, leaves of C.

galaticus have been used as laxative, cholagogue, anthelminthic (BAYTOP [2]; YEŞIL [3])

animal fodder (ERTUG [4]) and cooked food among the people (YEŞIL [3]). Poultices

obtained from the flowers (TUZLACI and DOGAN [5]) and mouthwashes obtained with

infusion of C. galaticus (ALTUNDAG and OZTURK [6]) have been used in the treatment of

toothache in folk medicine. Decoction obtained from the roots of C. galaticus is used because

of the purgative effect (OZTURK and OLCUCU [7]). Threat category of the endemic C.

galaticus is evaluated as LC (Least concern) (BOCUK & al. [8]; YILDIZTUGAY & al. [9]).

Antibacterial (TURKER & al. [10]; TURKER and KOYLUOGLU [11]), antitumor

(TURKER and KOYLUOGLU [11]) and anticancer (TOKGUN & al. [12]; KARAKAS & al.

[13]) activities of C. galaticus have been reported. Antioxidant activities of some species of

Convolvulus (C. arvensis, C. microphyllus, C. hystrix and C. dorycnium) were studied and it

was found that high phenolic content of these species led to strong antioxidant activities

(AWAAD & al. [14]; NACEF & al. [15]; DONIA & al. [16]; JAIN & al. [17]).

Grizzle bindweed is a valuable medicinal herb, but there are no reports on an in vitro

culture protocol of this species. The present work reports an in vitro culture procedure for

rapid clonal propagation of C. galaticus. Antioxidant activities and phenolic contents of field-

grown and in vitro-grown plant materials were also determined and compared by LC-MS/MS

method for the first time.

2.Materials and Methods 2.1 Clonal propagation

In vitro regeneration of C. galaticus was attempted by using two different explant

sources (field-grown plants and in vitro regenerated seedlings). Field-grown plant parts

(leaves and stems) were collected from AIBU Campus, Bolu, Turkey. Identification of the

species was made by using “Flora of Turkey and The East Aegean Island” (DAVIS [1]) and

voucher specimens (AUT-2026) were deposited at Abant Izzet Baysal University (AIBU)

Herbarium, Bolu/Turkey. Field-grown plant parts were washed 2 hours under running water

and then kept in sterile distilled water containing tween 20 (10 drops in 100 ml water) for 15

minutes. Surface sterilization was achieved in 0.1 % mercuric chloride (HgCl2) for 15 min

and then 70 % ethanol (EtOH) for 2 minutes. Plant parts were rinsed with sterile distilled

water 5 times. After surface sterilization of the stems and leaves, leaf, stem internode and

stem node explants were excised and placed in sterile disposable petri plates (80 x 15 mm)

containing 15 ml of Murashige and Skoog medium (4.43 g/l, MS, Sigma Chemical Co., St.

Louis, MO, USA) (MURASHIGE and SKOOG [18]), 30 g/l sucrose, 8 g/l Difco Bacto-agar

(pH 5.7, autoclaved for 20 min at 121°C and 105 kPa) with different combinations and

concentrations of plant growth regulators. In the second part of the experiment, in vitro

regenerated plantlets from stem node explant were used as donor plants. Three different

explants (leaf, stem internode and stem node) were excised and placed on MS medium with

different combinations and concentrations of plant growth regulators. All cultures were

incubated at 22 °C under a 16-h photoperiod (cool-white fluorescent lights, 22-28 µmol m-2

s-

1). After 6-8 weeks, regenerated explants were transferred to Magenta containers (GA-7

Vessel, Sigma Chemical Co.) containing MS medium with 1 mg/l gibberellin (GA3) for shoot

elongation for an additional two weeks. The shoot number and percentage of explants

producing shoots were recorded after 10 weeks for all explants. Tests had 10 replications for

each explant and the experiment was repeated three times.

Shoots were then separated individually and placed in rooting medium containing MS

including 1 mg/l GA3 and varying concentrations of different auxins. After 6 weeks, the

number of roots and percentage of explants producing roots were recorded. There were 10

replications and experiment was replicated three times. Rooted explants were transferred to

vermiculate (Agrekal®) in Magenta containers for acclimatization and after 3-4 weeks they

were transferred to plastic pots containing potting soil.

All data were analyzed by analysis of variance (ANOVA) and mean values were

compared with Duncan’s Multiple Range Tests using SPSS vers. 15 (SPSS Inc, Chicago, IL,

USA).

2.2 Plant material and extraction

Two different sources of the plant (field-grown and in vitro-grown) were used for

extractions. All plant materials were dried in a room avoiding sun light and then ground into

a powder. Field-grown aerial parts and in vitro grown seedlings of C. galaticus were

extracted with methanol. In methanol extraction, 20 g of each plant sample were soxhlet

extracted with 300 ml of methanol at 65 ºC for 12 hours and then filtered. Filtrates were

evaporated under vacuum using rotary evaporator to give the crude extracts.

2.3 Antioxidant assay

2.3.1Free radical scavenging activity

Free radical scavenging activity of methanolic extracts of C. galaticus was determined

spectrophotometrically by monitoring the disappearance of 2,2-diphenyl-1-picrylhydrazil

(DPPH, Sigma-Aldrich Chemie, Steinheim, Germany) at 517 nm, according to the method

described by BRAND-WILLIAMS & al. [19].

2.3.2 Determination of total phenolics content The phenolic contents in methanolic extracts of C. galaticus were determined

according to the procedure described by SLINKARD and SINGLETON [20] with the slight

modification of using a Folin-Ciocalteu phenolic reagent.

2.3.3 Determination of total flavonoid

The amount of total flavonoids in methanolic extracts was measured by aluminum

chloride (AlCl3) colorimetric assay according to the procedure described by MARINOVA &

al. [21].

2.4 LC-ESI-MS/MS analysis of the selected phenolics Analysis was performed on a Triple Quadrupole LC-MS system with an Agilent 1290

Infinity LC (Agilent) in Central Laboratory, METU, Ankara, Turkey. Triple Quadrupole LC-

MS system is equipped with a Jet Stream ESI source. Compounds were separated on an

Zorbax SB-C18 column (Agilent, 50 mm x 2.1 mm; 1,8μm particle size). The mobile phase

consisted of: (A) water containing 0.05 % formic acid + 5 mM ammonium formate (v/v) and

(B) methanol. A stepwise gradient from 5 % to 95 % solvent B for 13 min was applied to run

the separation. 5 µl of each sample extract was injected and flow rate was 0.3 ml/min. The

column temperature was maintained at 35 °C.

3. Results and Discussion Although C. galaticus is a very valuable medicinal plant, there is no any study about

in vitro propagation of this plant. We therefore aimed to develop an in vitro culture protocol

for high frequency regeneration of grizzle bindweed. Two different donor plants (field-grown

plants and in vitro regenerated seedlings) were used as an explant source. In the first part of

the experiment, field-grown plant parts (leaves and stems) were collected, surface sterilized

and three different explants (leaf, stem internode and stem node) were taken. Explants were

cultured on MS medium containing TDZ in combination with IAA or IBA (Table 1); BA in

combination with IAA or NAA (Table 2); KIN in combination with IAA (data not provided).

Among used explants only stem node explant was successful for shoot regeneration. Best

shoot regeneration was obtained with stem node explant on medium containing 0.5 mg/l TDZ

and 1.0 mg/l IAA (15.0 shoots per explant; 84 % explants formed shoots). Although 1

mg/TDZ and 1 mg/l IBA combination was effective in terms of mean number of shoots (12

shoots), shoot frequency was very low (20 %) (Table 1). The regeneration efficiency was not

found to be high with BA and IAA combinations and better shoot proliferation was observed

when BA was used alone (1, 3 and 5 mg/l) with regard to the mean number of shoots. But,

percentage of explants forming shoots was not efficient with these concentrations (53 %, 60

% and 40 %, respectively) (Table 2). KIN and IAA combinations were not effective for shoot

regeneration (2.3-3.3 shoots per shooting explant) (data not provided). In the second part of

the experiment, in vitro grown seedlings were used as donor plant. Three different explants

(leaf, stem internode and stem node) were excised and cultured on medium containing TDZ

in combination with IAA or IBA; BA in combination with NAA (Table 3).

Table 1. Effects of TDZ in combination with IAA or IBA on shoot regeneration from field- grown plants as an

explant source. Means with the same letter within columns are not significantly different at P>0.05.

Best shoot proliferation was obtained with node explant with 1 mg/l TDZ and 0.5

mg/l IBA (23.7 shoots per explant; 60 % explants formed shoots) (Table 3, Figure 1A). But,

twofold increase of IBA concentration (from 0.5 to 1 mg/l) decreased the shoot number twice

(from 23.7 to 12.3 shoots) (Table 3). Regarding the shoot frequency, best combination was

0.5 mg/l TDZ and 0.5 mg/l IBA with 87 % explants forming shoots (11.6 shoots) (Table 3).

Shoot regeneration was not obtained with leaf and stem internode explants (Table 3). Control

treatments involving no plant growth regulators produced no shoots in all 3 explants for all

experiments. (Table 1, 2 and 3). Indirect organogenesis was observed for all experiments

because of callus formation before shoot development.

% explants

forming

shoots

% explants

forming

shoots

% explants

forming

shoots

Control - - - - - - - - -

TDZ (mg/l) IAA (mg/l)

0.05 0.0 - - - - - - 4.3 ± 0.3cde

20

0.05 0.1 - - - - - - - - -

0.05 0.5 - - - - - - 2.7 ± 0.3def

50

0.05 1.0 - - - - - - 3.0 ± 0.0def

50

0.1 0.0 - - - - - - 5.3 ± 0.3bcde

20

0.1 0.1 - - - - - - 2.0 ± 0.6ef

25

0.1 0.5 - - - - - - 3.3 ± 0.9def

33

0.1 1.0 - - - - - - 3.7 ± 0.6def

67

0.5 0.0 - - - - - - - - -

0.5 0.1 - - - - - - 5.7 ± 0.9bcde

33

0.5 0.5 - - - - - - - - -

0.5 1.0 - - - - - - 15.0 ± 3.1a

84

1.0 0.0 - - - - - - 2.3 ± 0.3def

25

1.0 0.1 - - - - - - 6.0 ± 1.5bcd

33

1.0 0.5 - - - - - - 7.7 ± 0.9bc

33

1.0 1.0 - - - - - - 8.0 ± 2.2b

50

TDZ (mg/l) IBA (mg/l)

0.5 0.1 - - - - - - 5.3 ± 2.0bcde

33

0.5 0.5 - - - - - - - - -

0.5 1.0 - - - - - - 8.7 ± 0.9b

25

1.0 0.1 - - - - - - - - -

1.0 0.5 - - - - - - - - -

1.0 1.0 - - - - - - 12.0 ± 1.2a

20

3.0 0.1 - - - - - - - - -

3.0 0.5 - - - - - - - - -

3.0 1.0 - - - - - - - - -

5.0 0.1 - - - - - - - - -

5.0 0.5 - - - - - - - - -

5.0 1.0 - - - - - - - - -

Mean number of

shoots per shooting

explant (±SE)

Mean number of

shoots per shooting

explant (±SE)

Mean number of

shoots per shooting

explant (±SE)

E X P L A N T S

Plant Growth Regulators Leaf Stem internode Stem node

Table 2.Effects of BA in combination with IAA or NAA on shoot regeneration from field- grown plants as an

explant source. Means with the same letter within columns are not significantly different at P>0.05.

Regenerated shoots were cultured on shoot elongation medium containing 1 mg/l GA3

for additional 2 weeks (Fig. 1B). After 2 weeks, regenerated shoots were separated

individually and cultured on MS medium including 1 mg/l GA3 and IAA, IBA, 2.4-D or

NAA. Root induction was not occurred on the control treatment to which no auxin was added

to the media. Of the different auxins investigated for rooting, 1 mg/l IBA was more efficient

in terms of mean number of roots (5.5 roots). Regarding the root frequency, 5 mg/l IBA was

superior with the greatest percentage of root formation (100 %) (Table 4, Figure 1 C). Root

development was observed in 6 weeks. Medium containing IAA concentrations were also

effective for root formation. Although 1 mg/l NAA caused root formation (4 roots),

increasing concentrations of NAA (3, 5 and 7 mg/l) severely inhibited shoot development.

2,4-D concentrations were not effective in root formation (Table 4). The rooted plants were

transferred to Magenta containers including vermiculate for acclimatization (Fig. 1D). After

3-4 weeks, they were transferred to soil and kept under growth room conditions (Fig. 1E and

F).

% explants

forming

shoots

% explants

forming

shoots

% explants

forming

shoots

Control - - - - - - - - -

BA (mg/l) IAA (mg/l)

0.5 0.0 - - - - - - 3.3 ± 0.3def

58

0.5 0.1 - - - - - - 5.0 ± 0.6def

33

0.5 0.5 - - - - - - 6.3 ± 0.3bcde

33

0.5 1.0 - - - - - - 5.3 ± 0.7def

33

1.0 0.0 - - - - - - 13.3 ± 3.1a

53

1.0 0.1 - - - - - - 5.7 ± 0.3cdef

67

1.0 0.5 - - - - - - 3.7 ± 1.5def

75

1.0 1.0 - - - - - - 7.3 ± 2.3bcde

75

3.0 0.0 - - - - - - 9.3 ± 0.9abcd

60

3.0 0.1 - - - - - - - - -

3.0 0.5 - - - - - - 11.3 ± 0.3abc

75

3.0 1.0 - - - - - - 5.7 ± 0.3cdef

67

5.0 0.0 - - - - - - 12.0 ± 0.6ab

40

5.0 0.1 - - - - - - 4.3 ± 0.9def

40

5.0 0.5 - - - - - - - - -

5.0 1.0 - - - - - - - - -

BA (mg/l) NAA (mg/l)

0.5 0.1 - - - - - - - - -

0.5 0.5 - - - - - - - - -

0.5 1.0 - - - - - - - - -

0.5 3.0 - - - - - - - - -

1.0 0.1 - - - - - - 5.0 ± 0.7def

75

1.0 0.5 - - - - - - 3.0 ± 0.6ef

71

1.0 1.0 - - - - - - 2.0 ± 0.0ef

67

1.0 3.0 - - - - - - 4.0 ± 0.6def

33

3.0 0.1 - - - - - - 7.5 ± 1.1bcde

67

3.0 0.5 - - - - - - - - -

3.0 1.0 - - - - - - - - -

3.0 3.0 - - - - - - - - -

5.0 0.1 - - - - - - - - -

5.0 0.5 - - - - - - - - -

5.0 1.0 - - - - - - - - -

5.0 3.0 - - - - - - - - -

Mean number of

shoots per shooting

explant (±SE)

Mean number of

shoots per shooting

explant (±SE)

Mean number of

shoots per shooting

explant (±SE)

E X P L A N T S

Plant Growth Regulators Leaf Stem internode Stem node

Table 3. Effects of TDZ in combination with IAA or IBA and BA in combination with NAA on shoot

regeneration from in vitro regenerated seedlings as an explant source. Means with the same letter within

columns are not significantly different at P>0.05.

Table 4. Effects of the tested auxins on root formation from regenerated shoots. Means with the same letter

within columns are not significantly different at P>0.05.

% explants

forming

shoots

% explants

forming

shoots

% explants

forming

shoots

Control - - - - - - - - -

TDZ (mg/l) IAA (mg/l)

0.5 0.0 - - - - - - - - -

0.5 0.5 - - - - - - 6.7 ± 0.3ef

67

0.5 1.0 - - - - - - 9.3 ± 0.3bcde

67

1.0 0.0 - - - - - - - - -

1.0 0.5 - - - - - - - - -

1.0 1.0 - - - - - - 5.3 ± 0.3f

75

TDZ (mg/l) IBA (mg/l)

0.5 0.5 - - - - - - 10.8 ± 1.6bcd

87

0.5 1.0 - - - - - - 10.7 ± 0.3bcd

25

1.0 0.5 - - - - - - 23.7 ± 0.3a

60

1.0 1.0 - - - - - - 12.3 ± 1.2b

57

BA (mg/l) NAA (mg/l)

1.0 0.5 - - - - - - 6.3 1.3ef

87

1.0 1.0 - - - - - - - - -

3.0 0.5 - - - - - - 11.6 0.8bc

87

3.0 1.0 - - - - - - - - -

5.0 0.5 - - - - - - 4.7 ± 0.3f

50

5.0 1.0 - - - - - - - - -

7.0 0.5 - - - - - - 8.3 ± 0.7cdef

63

7.0 1.0 - - - - - - 7.7 ± 0.3def

50

10.0 0.5 - - - - - - 6.3 ± 0.9ef

50

10.0 1.0 - - - - - - 6.3 ± 0.3ef

45

Mean number of

shoots per shooting

explant (±SE)

Mean number of

shoots per shooting

explant (±SE)

Mean number of

shoots per shooting

explant (±SE)

E X P L A N T S

Plant Growth Regulators Leaf Stem internode Stem node

Treatments % explants

forming roots

Control - - -

IAA (mg/l)

1.0 1.7 ± 0.3 bc

40

3.0 2.0 ± 0.4 bc

60

5.0 1.7 ± 0.3 bc

75

7.0 1.3 ± 0.3 bc

75

10.0 4.0 ± 0.0 ab

75

IBA (mg/l)

1.0 5.5 ± 1.6 a

80

3.0 3.8 ± 0.9 ab

83

5.0 3.2 ± 0.9 ab

100

7.0 2.3 ± 0.9 bc

75

10.0 3.5 ± 1.6 ab

80

NAA (mg/l)

1.0 4.0 ± 0.0 ab

33

3.0 - - -

5.0 - - -

7.0 -

2,4-D (mg/l)

0.5 - - -

1.0 - - -

Mean number of roots per

explant (±SE)

Only one investigation has been carried out on in vitro regeneration of members of the

genus Convolvulus. ABBAS & al. [22] reported the in vitro culture protocol of Convolvulus

scindicus Stocks. They obtained best shoot proliferation with 2.5 mg/l BA along with 0.5

mg/l KIN and 0.5 mg/l NAA. Similar to our results, they used nodal segments for

establishing in vitro cultures. They observed maximum number of roots (1.5) per explant and

maximum rooting frequency of 67 % with MS medium containing 2 mg/l IAA (ABBAS & al.

[22]). On the other hand, best root formation was obtained with 1, 3 and 5 mg/l IBA in our

study with rooting frequencies of 80, 83 and 100, respectively (Table 4).

Leaf and stem internode explant of C. galaticus were unsuccessful to develop

adventitious shoots. Shoot multiplication was obtained with stem node explant including

meristematic cells for this plant. Micropropagation of many medicinal plant species has been

achieved through different tissue culture techniques. In many cases, actively growing shoot-

tips or axillary buds, both of which already contain de nova primordia, were used as a starting

material (ROUT & al. [23]). This method remains the most widely used method in

commercial micropropagation and produces the most true-to-type plantlets (BROWN and

THORPE [24]; KANE [25]). It seems that the proliferative potential of meristematic cells of

the stem node explant including axillary bud in our study is readily stimulated by

exogenously added growth regulators, resulting in multiple shoot formation (KANE [25]).

Our findings indicated that TDZ was the most critical plant growth regulator for multiple

shoot formation from stem node segments when used in combination with IAA or IBA. A

possible synergism between TDZ and auxins, both endogenous and some of the exogenous

may lead to multiple shoot formation. The promoting effect of TDZ on in vitro development

has been lately reported for many species (HUETTEMAN and PREECE [26]; LU [27];

YILDIRIM and TURKER [28]). MURCH & al. [29] showed that the occurrence of

regenerants in TDZ-treated plants may be an adaptive reproductive mechanism to overcome

the imposed stress. MURTHY & al. [30] hypothesized that under the influence of TDZ, a

relatively high level of accumulation of minerals or other metabolites occurs in the tissues,

and this causes a stress in plants (explants). To overcome this physiological stress, the plant

tissue modifies its metabolic processes, resulting in the production and accumulation of

various metabolites and culminating in the formation of regenerants.

The scavenging activity of DPPH radical caused by antioxidants was determined by

measuring the decrease in its absorbance at 517 nm. Ascorbic acid was used as the

antioxidant standard in this experiment. In the present study, antioxidant activity of

methanolic extract of field-grown and in vitro-grown plants was assessed. The free radical

scavenging activity (DPPH), total phenolic content (Folin-Ciocalteau) and total flavonoid

content (aluminum chloride colorimetric) were used in this assessment. Methanolic extract of

field-grown C. galaticus showed better free radical scavenging activity than in vitro-grown

plants. Although over 50 % inhibition of DPPH was obtained at 50 μg/ml concentration with

field-grown C. galaticus, 100 μg/ml concentration was required for in vitro-grown C.

galaticus. The free radical scavenge tendency of both extracts increased when their

concentrations increased (Table 5). The best DPPH scavenging activity of field-grown and in

vitro-grown plants was obtained at 200 μg/ml concentrations (90.12 % and 92.75 %,

respectively) that they showed high active radical scavenge capability as much as ascorbic

acid (99.53 %) (Table 5).

Figure 1. In vitro regeneration of C. galaticus. (A) Shoot regeneration from stem node explant on medium

containing 1 mg/l TDZ and 0.5 mg/l IBA, (B) Shoot elongation of regenerated shoots on medium containing 1

mg/l GA3, (C) Rooting of the regenerated shoots on medium containing 3 and 5 mg/l IBA, (D) Regenerated

plant in magenta container including vermiculate for acclimatization, (E) Regenerated plants transferred to cups

containing sterile potting soil under high humidity conditions, (F) Regenerated plants transferred to cups

containing sterile potting soil under growth room conditions.

Table 5. % inhibition of DPPH by C. galaticus extracts.

Treatments 12.5 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml 200 µg/ml

Ascorbic acid 95.59 95.73 96.2 96.14 99.53

Field-grown C. galaticus 24.13 42.71 76.33 88.34 90.12

In vitro -grown C. galaticus 14.97 27.79 46.05 71.89 92.75

% Inhibition of DPPH

Concentrations

Methanol extract of field-grown plant contained higher phenolic (84.689 mg gallic

acid equivalent/g dried extract) and flavonoid (48.760 mg catechol acid equivalent/g dried

extract) content than in vitro-grown plant (43.573 mg gallic acid equivalent/g dried extract,

30.110 mg catechol acid equivalent/g dried extract, respectively ) (Table 6). Hydroxyl groups

on phenolic compounds have scavenging ability so they are very important plant constituents.

A number of studies reported a significant relationship between the phenolic contents of plant

extracts and their antioxidant properties (GULCIN & al. [31]). Field-grown leaves of C.

galaticus had higher phenolic and flavonoid content thereby having higher DPPH scavenging

activity (Table 5 and 6). Numerous studies have revealed that environmental stress often raise

the accumulation of the phenolics, which are believed that to play a regulatory role in some

metabolic process (DIXON and PAIVA [32]; SOLECKA [33]; JANAS & al. [34]).

THAKRAL & al. [35] reported the antioxidant activity of aerial parts of Convolvulus

arvensis by DPPH method. Similar to our results, C. arvensis extracts showed dose

dependent free radical scavenging property. THAKRAL & al. [35] showed that 50 % DPPH

inhibition was observed at 131.03 µg/ml concentration with methanolic extract of C.

arvensis. On the other hand, 50 % DPPH inhibition was obtained at 25-50 µg/ml

concentrations with field-grown C. galaticus and at 50-100 µg/ml concentrations with in

vitro-grown C. galaticus in our study (Table 5). JAIN & al. [17] reported that methanol

extract of Convolvulus microphyllus showed 50 % DPPH inhibition at 75 µg/ml

concentration with DPPH method. Ethyl acetate and n-butanol fractions of Convolvulus

dorycnium leaves showed 50 % DPPH inhibition at 3.2 µg/ml and 6.9 µg/ml, respectively

(NACEF et al. [15]).

Table 6.Total phenolic and flavonoid content of C. galaticus extract.

Methanolic extracts of field-grown and in vitro-grown C. galaticus were subjected to

Liquid Chromotography-Tandem Mass Spectrometry analyses and results were summarized

in Table 7. The chromatogram of phenolic standards (each standard, 5 ppm in mixture) was

obtained via gradient methanol flow. The phenolic content of the methanolic extracts was

compared with their standard chromatograms and identified with mass spectrometer (MS).

Finally, quantity of each phenolic compound in the extract was determined (Table 7).

According to LC-ESI-MS/MS results, the amounts of studied phenolic compounds in

field-grown plant were higher than those in in vitro-grown plant (Table 7). Concentrations of

phenolic compounds in field-grown plant extract were at least 3 times higher than the other

extract. Caffeic acid was found in field-grown plant nearly 10 times higher (157.432 µg/g)

than in vitro-grown plant (16.464 µg/g). Similarly, rutin was found 28 times higher (286.9

µg/g) than in vitro grown plant (10.45 µg/g). Although rutin was dominant compound in

methanol extracts of field-grown leaves, caffeic acid was dominant in in vitro-grown leaves.

Methanol extracts of field-grown plant contained from the highest to lowest amount rutin

TreatmentsTotal Phenolics in mg GA/g dry

extract

Total Flavonoids mg CE/g dry

extract

Field-grown C. galaticus 84.689 ± 0.000 48.760 ± 0.001

In vitro -grown C. galaticus 43.573 ± 0.000 30.110 ± 0.001

(286.9 µg/g), caffeic acid (157.432 µg/g), coumarin (15.382 µg/g), kaempferol (14.832 µg/g),

vanillic acid (6.264 µg/g), coumaric acid (4.01 µg/g) and epigallocatechin (0.094 µg/g).

Methanol extracts of in vitro-grown plant contained from highest to lowest amount caffeic

acid (16.464 µg/g), rutin (10.45 µg/g), vanillic acid (2.37 µg/g) and coumaric acid (0.204

µg/g). The reason of high phenolic content of field-grown plant may be due to the stress

conditions in the natural environment (MICHALAK & al. [36]). When plants are exposed to

different types of stress, such as drought, heat, ultraviolet light, air pollution, and pathogen

attack, the synthesis of some phenolic compounds is induced adapting to these stresses

(RIVERO& al. [37]).

AL-RIFAI & al. [38] determined the flavonoids (kaempferol and quercetin) in

methanolic extract of Convolvulus pilosellifolius Desr and kaempferol was more abundant

than quercetin in C. pilosellifolius. Quantity of quercetin and kaempferol was found as 4.27

µg/ml extract and 6.14 µg/ml extract, respectively. Similarly, kaempferol was found more

than quercetin in our study. On the other hand, quantity of kaempferol was found as 14.832

µg/g and quercetin was lower than 0.01 µg/g in our study (Table 7).

Table 7. Identified phenolic compounds and their amounts in the methanolic extracts of field-grown plant and in

vitro-grown plant of C. galaticus. “a” indicates peaks for standards having the same-close retention times.

Field-grown C. galaticus In vitro -grown C. galaticus

Gallic acid monohydrate 1 0.92 ≤ 0.01 ≤ 0.01

Pyrocatechol 2* 1.902 ≤ 0.01 ≤ 0.01

Procyanidin B1 2.225 ≤ 0.05 ≤ 0.05

(-) epigallocatechin 3* 2.497 0.094 ± 0.006 ≤ 0.01

(+) catechin 2.539 ≤ 0.01 ≤ 0.01

Procyanidin B2 4 2.886 ≤ 0.05 ≤ 0.05

Vanillic acid 5* 3.063 6.264 ± 0.26 2.37 ± 0.112

Caffeic acid 3.092 157.432 ± 0.998 16.464 ± 0.166

Procyanidin C1 6* 3.216 ≤ 0.5 ≤ 0.5

(-) epicatechin 3.32 ≤ 0.01 ≤ 0.01

p-coumaric acid 7 3.884 4.01 ± 0.168 0.204 ± 0.004

(±) Taxifolin hydrate 8 4.07 ≤ 0.01 ≤ 0.01

Coumarin 4.716 15.382 ± 0.442 ≤ 0.025

Luteolin-7-O-β-D glucoside 9* 4.809 ≤ 0.025 ≤ 0.025

Rutin hydrate 4.898 286.9 ± 2.222 10.45 ± 0.46

Resveratrol 4.956 ≤ 0.01 ≤ 0.01

Myricetin 10 5.27 ≤ 0.01 ≤ 0.01

Kaempferol 11 5.42 14.832 ± 0.124 ≤ 0.01

Daidzein 12 5.829 ≤ 0.01 ≤ 0.01

Quercetin 13 6.073 ≤ 0.01 ≤ 0.01

Genistein 14 6.425 ≤ 0.01 ≤ 0.01

Apigenin 15 6.945 ≤ 0.01 ≤ 0.01

STANDART COMPOUNDS Retention time

(min)

EXTRACTS (µg/g of dry extract)Peak number

4. Conclusion This paper, as being the first report, described an efficient and rapid regeneration

system for C. galaticus, an endemic plant. Plant tissue culture is an alternative method of

commercial propagation and is being widely used for the commercial propagation of a large

number of plant species, including many medicinal plants (ROUT & al. [23]). It is believed

that this protocol will have an important contribution for in vitro conservation and mass

propagation of this endemic plant. Furthermore, phenolic constituents and antioxidant activity

of this plant was revealed for the first time with this study. Comparison between in vitro-

grown and field-grown plants in terms of their phenolic constituents and antioxidant activities

was performed revealing the quality of in vitro-grown plants. C. galaticus contained the

considerable amounts of phenolic compounds, such as rutin and caffeic acid. Considering the

strong biological activity of phenolic compounds, future studies should be focused to increase

the amount of phenolics in in vitro-grown plant parts by applying different stress conditions.

5. Acknowledgements The authors are grateful to The Scientific and Technological Research Council of

Turkey (TUBITAK) for financial support (TBAG-HD-211T172).

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